IAA which subsequently led to the detection

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NATURALLY OCCURRING HORMONES IN

TROPICAL PLANTS*

BY S. M. SIRCAR

Botany Department, Calcutta University, Calcuttt< -19

IT is a great honour to be elected as the President of the Indian Society for Plant Physiology. I feel very embarrassing to take the chair which in the past was adorned by several distinguished plant physiologists of India. There may be some shortcomings in fulfilling the responsi-bilities of this high office but, I am sure, the members who were gracious enough to elect me will also forgive my failings.

India today stands at the threshold of improving the standard of living of her increasing population. To politicians and scientists this primarily rests on how the country could be made self-sufficient in food production. In this context great responsibilities lie with the botanists more particularly with those who are concerned with the physiology of tropical plants to add new knowledge for practical application and solve problems impeding production of more food from the available land. I am sure, the present generation of plant physiologists wiil face this task with great enthusiasm. Plant physio-logy today has practically merged with plant biochemistry and bio-physics. Researches in these disciplines are progressing at a very rapid rate with the help of new techniques and advanced physical instruments. Many of us are often faced with difficulties for not having enough of imported equipments and this often disheartens the enthusiasm for research. Pending the availability of foreign exchange to buy these imported equipments or their manufacture in India, the situation of dearth of these equipments may be viewed in a different perspective. May I recall here that plant physiology had a very good beginning and built up a tradition of researches in India with the help of delicate and precision instruments invented by Sir J. C. Bose? I do hope that the example and tradition left over by our distinguished scientist will stimulate others to overcome some of their difficulties in experimentation by devising new techniques and appliances. J t needs no emphasis that the whole nation looks ahead for your earnest efforts in solving the difficulties beset with improvement of agricultural production.

With these observations I would like to present a review of research work in which my colleagues and myself in India have been engaged for the past decade and a half.

;c.

76

Till the middle of this century, IAA has been considered as the only naturally occurring auxin in plants. With the application of modern techniques, particularly adsorption and partition rapn chromatography, the picture tended to show a marked change in the concept of natural auxin. To this is added the discovery of GA and the interest of the Japanese Scientists created on the similar and dissimilar properties of this fungal product with IAA which subsequently led to the detection of GA in higher plants. The belief of the occurrence of natural auxins other than IAA gained ground when several indole compounds were identified from plants by chromatography. The different indole com-pounds present in the growing tissue ar" suggestive of their functional importance and have shown growth response when applied to tc:st materials. Some of the more important compounds are: indoleaceto-nitrile, indoleacetaldehyde, indolepyruvic acid. Bentley (1958) has reviewed the indole compounds reported L; be present in different plant materials (3-indolecarboxylic acid, indole ethyl acetate, indolealdehyde, indole and N-methyl tryptophan, 5-hydroxy N, N-dimethyl tryptamine, 5-hydroxy tryptamine, 5-hydroxy IAA and skatole). The question arises whether these are products of biogenesis of IAA or degradation of tryptophan in the metabolic reactions of tissue with no significance as auxin affecting growth. The presence of auxin other than IAA is emphasized by her on the basis of following experimental evidence: (i) in many plants several workers failed to detect IAA after application of chromatography with different solvents; (ii) concentration of IAA is often very small in comparison to total auxin or it is not operating at certain stages of the development of a plant; and (iii) the presence of growth-promoting zones in the chromatogram at Rf differwt from IAA which are yet unidentified and may be other than indole com-pounds. Besides these ether-soluble indole compounds, Bennet·Ciarke

eta!. (1953) have reported the presence of an ether-soluble unidentified auxin from several plant materials and have termed it as a-accelerator which shows weak activity. Bentley (1958) has remarked that it may be the same as 'W ', a water-soluble substance extracted by her from cabbage, and is not free auxin as its presence is detected only after treatment with heat and alkali.

\

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HORMONES IN TROPICAL PLANTS 77

his co-workers (1955; 1956) have observed the formation of indole-aspartic acid when IAA is fed to a wide variety of tissues. It is water-soluble and sparingly water-soluble in ether. The question arises whether it is a physiologically active auxin or a detoxication prcduct of IAA like a similar conjugate, indole acetyl-L-aglutamine formed in human system. Street (1963), however, has noted it as a growth regulators. Ascorbigen is another auxin protein complex discovered by Prochazka

et al. (1956; 1957), ·Bose and Guha (1959) and Guha (1961), Malakar

and Guha (1961). It yields IAA on alkali hydrolysis and ascorbic acid (AA) on acid hydrolysis. The presence of ascorbigm in different organs of the plant is presumed to have significance in growth processes. The growth-promoting effects of ascorbic acid on wheat has been reported by Chinoy eta!. (1958; 1961). Their results indicate that in dry excised embryos of Arachis hypogea the acceleration of growth synchronizes with an enhanced biosynthesis of ascorbic acid (AA). They have further concluded that early and more rapid biosynthesis of AA by growing plants is significant in growth and flowering of plants. Thus the free indole auxins are not the only physiologically active ones and the auxin bound with a proteinaceous compound is not only the storage reserve but also actively concerned in the growth process.

Reviewing briefly the whole position of the occurrence of natural auxins it is reasonably clear that in addition to ether-soluble indole auxins there exists in tissues aquec:us ether-insoluble auxins -whose precise chemical identification has not yet been possible because of their very small amounts. Further investigation is needed to determine precisely whether these are true auxins acting by themselves or form compounds with indoles or interact with TAA for physiological activity.

Researches on the occurrence of natural auxins by Indian workers are not many due to dearth of technical personnel, equipment, encourage-ment and proper perspective in scientific research. The main concern has been to obtain spectacular results of practical importance in agri-culture and hortiagri-culture with little enthusiasm for probing into the fundamental issue of the nature of auxins present or to elucidate how applied auxins interact with natural ones.

Early in 1948-50, Asana, Sircar and Uttaman reported the growth-promoting effects of plant extracts, but the results failed to create interest for lack of chemical characterisation of the extracts.

78

thicker. They tentatively concluded that the growth-inhibiting sub-stance in the extract was free auxin. Except the two varieties, other varieties did not show the presence cf the inhibiting substance in the extract prepared by soaking onl}. But when the extract was prepared by puncturing the soaked seeds near the embryo the inhibiting sub-stance came out showing that it did not diffuse out through the seed-coat in other varieties. That the inhibting effect was not due to salts was ascertained by testing the ashed extract. While investigating the effect of coconut water on the growth .of immature embryos of maize Uttaman (1949) noted that coconut water has a decisive depressing effect on the growth of the embryo and that the effect on the root is more pronounced than on the shoot. In a later paper he reported the comparative effects of coconut water with corn germ extract (Uttaman, 1950). The effect of the corn germ extract on the growth of maize embryo is more marked than that of the coconut water and is of more progressive character. The results show that the growth factor in coconut water is different from that of corn kernel. The chemical nature of the corn germ extract or coconut water factor was not investigated. The growth-promoting properties of rice washings on 7 varieties of sugarcane were reported by Vijayasardhy (1952), but the actual chemical responsible for this was not identified. Sircar and his associates made the first attempt in this country to determine the IAA content of the rice grains by A vena coleoptile curvature test for which the technique and the apparatus used have been described elsewhere (Sircar and Das, 1950; 1951). The results show 0·17 million in water and 0· 50 million T.D.C. in alkali extract per gm. of dry endosperm.

'

HORMONES IN TROPICAL PLANTS _i9

80 S. M. SIRCAR

carpel is much greater than that of the endosperm, but the total auxin is more than twice that of the young carpel. In the young embryo the total auxin is much less than that of the endosperm, but the con-centration is more or less the same in both these parts. Husks in the early stages of development showed some auxin which decreased gra-dually and 7 days after anthesis practically nothing was left in them. These results indicate that the internal auxin concentration largely regulates the growth of various organs of the rice plant.

Sircar and Kundu (1960) estimated free auxins at three stages of the rice plant growing in nutrient solutions with added growth sub-stances, IAA, NAA and MH. The increase in auxin level by the external application of the growth substances was regularly noted in all the stages of the plant. They ·have observed that the use of root inhibition technique for the assay of auxin from plant materials is of limited value, nevertheless from the comparative results of the auxin levels of the organs from the treated and untreated plants an increase in the endogenous auxin concentration after treatments with growth substances is evident. The earlier observation of Sircar (1958) on the presence of high auxin level at the shoot apex at the time of tran-sition was confirmed which would suggest that auxin content was not reduced prior to flowering but it accelerated flowering by the elongation of the shoot apex. Auxin tends to accumulate in the apex after trans-port from the leaves and stem where lower values are recorded at later stages of growth. The external supply also increased the free auxin content of the root which stimulated both root elongation and number. The endogenous a1;xin contents of the root, stem and leaf were lower in NAA and MH than in IAA. The high level after treatment with IAA is obviously related to the uptake of IAA by plant tissue. They have suggested that the accumulation takes place in some form from which it can be readily made available for growth processes and does not cause toxic effect as high auxin level in the root is not reflected by decrease in growth, but rather by stimulation in length and number. The high auxin level after treatment with NAA and MH has been inter-preted as a sort of competitive action between natural lAA and MH

HORMONES IN TROPICAL PLANTS 81

and extending stem of petkus winter rye in relation to vernalization and daylength were studied by Das (1958) by root inhibition test. There was no appreciable change in the auxin content in the apex, either before or immediately after floral initiation. Irrespective of vernalization and photoperiodic treatments and time for planting, the amount of auxin in the plant is approximately the same at the same stage of floral development. The auxin content of the nodes and internodes bear a clear relationship to the extension growth of the internodes.

The significance of the movement of flowering substances in the leaves of mango (Mangifera indica L.) has been shown by Singh

eta!. (1959). Young seedlings were made to flower and fruit when

grafted to a comparable shoot of bearing tree and given an additional treatment of defoliation along with the gildling ofthe scion shoot below the union. It is suggested that the leaves on seedling stocks may produce sufficient auxin to cause an adverse balance between auxin and the hormone which induces flowering.

Nagarajan and Gopalkrishnan (1958) reported the presence of root-inducing substances in groundnut seeds. Seeds of spreading dormant variety (TMV3) of groundnut were soaked in aqueous extract prepared from seeds of non-dormant bunch variety (TMV2) and germinated in sand with a view to study the effect of the water extract on breaking of dormancy. The seedlings that germinated showed an interesting phenomenon of adventitious root formation from the cotyledonary bases and stalks. These roots were di or triarch in T.S. No such incidence was found in the seeds soaked in water. Rajan (1958) observed that bioassay of cattle urine shows the presence of the significant amount of substances which have remarkable influence in regulating plant growth processes.

82 S. M. STRCAR

bioassay of growth substances; chemical identification was sought from the Rf of chromatograms with different solvents. In the neutral ether fraction no growth-promoting substance was detected either by bioassay or spot colour reaction. The acid ether fraction showed significant growth promotion in bioassay and the colour reactions of the chromatograms run with iso propanol-ammonia-water were noticed at Rf0·73-0·75, 0·76 and 0·91-0·99. It was confirmed that the growth substance at Rf 0·73-0·75 was fAA. The growth factor at Rf 0·91-0·99 appeared to be an indole compound as it gave spot colour reac-tions with Salkowski and Ehrlich reagents. The ether-insoluble or aqueous fraction showed marked promotion of growth at three zones: Rf 0·12-0·25, 0·37-0·62 and 0·75-1·0. Inhibition of root growth was also observed at Rf 0 · 62-0 · 75. Positive Salkowski and Ehrlich reactions were given by the fractions with Rf 0·71-0·75 and 0·97-0·99 suggesting the presence of indole compounds. It is interesting to note that the substance present in the fraction with Rf 0 · 49-0 · 60 has marked growth-promoting activities both in the root and shoot of the test material. It gives a colour reaction with potassium permanganate like GA but at widely different Rf. On the basis of these results Sircar and Roy (1961) concluded that the root extract of water hyacinth contains growth substances in addition to indole compounds or gib-berellins. Cell growth and metabolism influenced by the presence of naturally occurring growth substances in the root extract of water hyacinth has been studied by Ganguly and Sircar (1964). The active substance from chromatogram elutes of the ether-insoluble water fraction of the extract show the elongation of the cortical cells from different internodes of germinating pea seeds. The number of cells in the shoot tip measured by haemocytometer and the frequency of cell division in the root tip are also increased. The rate of cambial activity is accelerated and the diameter of the xylem vessels in the treated shoot is significantly larger than the control. Associated with these changes in the anatomical features of the pea seedlings a large-scale mobilization of sugar and nitrogenous constituents from coty-ledons to different internodes is noted. That the metabolic efficiencv

HORMONES IN TROPICAL PLANTS ₈₃

in the root tissue presumably associated with the seasonal variation in growth activities of the plant.

Maheshwari, Johri and Bhalla (1964) have studied acidic auxins in the maturing ovules of cotton (Gossypium hirsutum var. Indore 2).

The analysis of ovules at six different stages of development for the auxins in the acidic fraction show two spots of activity on the A vena mesocotyl sections; one with Rf 0 · 69 coinciding with that of synthetic IAA and the other unidentified spot at Rf 0 · 36 with isopropanol-ammo-nia water as solvent. The concentration of auxin is maximal just after pollination and minimal at maturity. Twenty-two days after pollination a small but unmistakable rise of the auxin content is evident which coincides with the differentiation of the embryo. The peak value per ovule is attained in the middle of the ripening period of the seed.

The presence of leucoanthocyanin in Diospyrus embryopteris and

palmyra palm (Borassus flabellifer) by chemical tests has been reported by

Sircar and his colleagues but the growth-promoting activity has not yet been confirmed by carrot root test of Steward (Sircar eta!., 1965,

unpub-lished report). The presence of ether-soluble free IAA was detected from the cellular endosperm. It gives positive growth promotion in bioassay in wheat coleoptile test and is positive in Ehrlich reaction and has characteristic fluorescence under ultra-violet light. In the liquid endos-perm (milk) bioassay and fluorescence tests indicate the presence of IAA, but no positive colour reaction could be noted when chemical tests were performed with the developed chromatogram. The water-soluble bound auxin precursor was ide!ltified from the cellular as well as liquid endosperm. The aqueous portwn after removal of any free auxin by ether was hydrolysed at pH 9 · 5 for 10 minutes, with the hydrolysep fraction it gave positive growth promotion in bioassay tests and has characteristic fluorescence under ultra-violet light, it is Ehrlich positive and has as Rf identical with that of IAA. It appears that the pro-portion of free to the bound form or precursor of auxin is more in the young than in the older fruits, the liquid (milk) portion being the richer source than the cellular (meat) portion. With maturitv how-ever, which results in the gradual transformation of the liquid' to the cellular endosperm, the level of the free form of auxin decreases and that of the bound for~ i!lcreases, the c~llular e~dosperm now being the richer source. Prehmmary study With Cass1a fistula which has

long cylind.rical pod~ s~~w the presence ~f f~e~ IAA in the seeds of mature fruits. An Inhibitor, presumably mhibJtor B and gibberellins have also been indicated but they need further confirmation with othe; bioassays and chemical tests.

REFERENCES

ANDREAE, W. A. AND Gooo, N. E. (1955). The formation of indoleacetylaspartic

acid in pea seedlings. Plant Physiol., 30, 380-82.

- AJ'!D VAN YssELSTEIN, M. W. I;J. (1956): St1:1dies on 3-indoleacetic acid

-84 S. M. SIRCAR

ASANA, R. D. AND MANI, V. S. (1948). On some physiological properties of extracts

of wheat grains. Curr. Sci., 17, 360-62.

BENNET CLARKE, T., TAMBIAH, M. S. AND KEFFORD, N. P. (1953). Estimation of

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BENTLEY, J. A. (1958). The naturally occurringauxins and inhibitors. Ann. Rev.

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BosE, S. AND GuHA, B. C. (1959). Ascorbigen in plant materials. Sci. and Cult.,

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CHINOY, J. J., GARG, 0. P. AND NANDA, K. K. (1958). Interaction of ascorbic

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- - , GROVER, R. AND NANDA, K. K. (1961). Role of sucrose in the growth reaction

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FRANSSON, P. (1959). Studies on a shoot and root elongation stimulator in Pinus

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GUHA, B. C. (1961). Studies on ascorbigen. Jour. Chem. Soc., 38, 492-94.

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Exp. Bioi., 2, 198-202.

MALAKAR, M. C. AND GUHA, B. C. (1961). Isolation of bound ascorbic acid

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MuKHERJEE, R. K., BHANJA, A., RoY BURMAN, P. AND SIRCAR, S. M. (1964).

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SINGH, K.

J:'.,

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HORMONES IN TROPICAL PLANTS ₈₅

SIRCAR, S. M. AND DAs, T. M. (1950). A chamber for phytohormone researches in the tropics. Sci. and Cult., 15, 282-84.

(1951). Growth hormone in rice grains germinated at different

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- - - . (1954). Studies in the physiology of rice IX. Auxin content of the

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SrRcAR, S. M. AND KUNDU, M. (1959). Effect of root extract of water hyacinth on the growth and flowering of rice. Sci. and Cult., 24, 332-33.

- - - - . (1960). Growth-regulating properties of the root extract of water

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